The immune system comprises a complex array of cellular and molecular mechanisms which recognizes and targets pathogenic microorganism or the cells infected with them. Recently, MBL has gained great interest as an important part of the innate immune system, that is, the immune system which at time of birth is operational, in contrast to the adaptive immune defence which only during infancy obtains its full power of protecting the body (Janeway et al. 1999). Upon binding of carbohydrates of microbial surfaces, MBL mediates activation of the complement cascade, a series of enzymatic activation steps, which eventually label the target for destruction by phagocytosis or by lysis of the microorganism (Law & Reid, 1995). The complement system is being activated through at least three distinct pathways, designated the classical pathway, the alternative pathway, and the MBLectin pathway (Janeway et al., 1999). The classical pathway is initiated when complement factor 1 (C1) recognizes surface-bound immunoglobulin. The C1 complex is composed of two proteolytic enzymes, C1r and C1s, and a non-enzymatic part, C1q, which contains immunoglobulin-recognizing domains. C1q and MBL shares structural features, both molecules having a bouquet-like appearance when visualized by electron microscopy. Also, like C1q, MBL is found in complex with two proteolytic enzymes, the mannan-binding lectin associated proteases (MASP). The three pathways all generate a convertase of complement factor 3 (C3 convertase) bound to the surface of the activating surface, i.e., the targeted microbial pathogen. Conversion of C3 into surface bound C3b is pivotal in the process of eliminating the microbial pathogen by phagocytosis or lysis (Janeway et al., 1999).
Mannan-binding lectin (MBL), also named mannan-binding protein or mannose-binding protein (MBP), was first characterized in rabbits (Kawasaki at al., 1978). Mannan-binding lectin (MBL) belongs to a group of soluble Ca2+-dependent (C-type) lectins containing a C-terminal carbohydrate recognition domain and a collagen-like region characterized by repeated triplet-motifs of glycine (Gly) followed by two non-glycine amino acids. Thus, MBL belongs to the group of collectins, i.e., C-type lectins with collagen-like regions, which in addition comprises the lung surfactant proteins A and D as well conglutinin and CL-43, these, however, only having been characterized in cattle (Holmskov et al., 1994). The human MBL protein is composed of up to 18 identical 32 kDa polypeptide chains (Lu et al., 1990), each comprising a short N-terminal segment of 21 amino acids including three cysteine residues, followed by 7 repeats of the collagenous motif Gly-X-Y interrupted by a Gin residues followed by another 12 Gly-X-Y repeats. A small 34 residue ‘neck-region’ joins the C-terminal Ca2+-dependant lectin domain of 93 amino acids with the collagenous part of the molecule (Sastry et al., 1989). Three MBL polypeptide chains are joined in a MBL subunit. MBL consists of up to six 15 nm stalks of MBL subunits joined at the base of the bouquet. Later work characterized MBL in rodents (Mizuno et al., 1981; Oka et al., 1988), cattle (Holmskov et al., 1993a; Kawai et al., 1997), and chicken (Oka et al., 1985; Laursen et al., 1995; Laursen et al., 1998). In rodents (Drickamer et al., 1986) and rhesus monkeys (Mogues et al., 1996) two types of MBL genes have been identified, usually designated as A and C forms. In rats, an MBL ‘B’ pseudogene was found (Drickamer et al., 1985) and recently an MBL pseudogene was also cloned from the human genome, sequence analysis suggesting this to be remnants of a primate MBL-A gene (Guo et al., 1996). Human MBL was characterized by Kawasaki et al., in 1983. Only one active human MBL gene has been identified, comprising four exons with three intervening introns of the MBL gene spanning approximately 6 kb and is located at 10q11.2-q21 (Sastry et al., 1989; Taylor et al., 1989).
The collagenous regions of the three polypeptide chains combine to form a subunit which is stabilized covalently by disulphide bridges. Individual subunits are joined by disulphide bridges as well as by non-covalently interactions (Lu et al., 1990).
The position of these disulphide bridges has, however, not been fully resolved. SDS-PAGE analysis under non-reducing conditions of MBL shows bands with an apparent molecular weight (m.w.) larger than 200 kDa presumably representing blocks of 3, 4, 5 and even 6 assembled subunits (Lu et al., 1990).
The actual number of subunits in the natural human MOL protein has been controversial. Lipscombe et al, (1995) obtained data by use of ultracentrifugation suggesting 25% of human serum MBL to be made of 2-3 subunits and only a minor fraction reaching the size of 6 subunits. The relative quantification was carried out by densitometry of Western blots developed by chemiluminescence. Lower efficiency in transferring high molecular weight protein onto membranes compared to proteins of lower molecular weight make analysis through this methodology complicated. Lu et al. (1990) found by SDS-PAGE analysis of fractions from ion exchange chromatography that the predominant species of covalently linked MBL subunit chains consisted of tetramers while only pentameric or hexameric complexes activated complement. Gel permeation chromatography (GPC) analysis, in contrast, suggests that MBL is comparable in size with the C1 complex. GPC can be carried out under conditions which allow for a study of the importance of weak protein-protein interactions in the formation of MBL molecules and, in combination with standard MBL assay techniques, also allows for unbiased determination of the MBL content in the GPC fractions.
The in vivo role of MBL seems mainly to relate to the innate immune system as a humoral factor mediating some anti-microbial activity, which does not require maturation into self/non-self discrimination like the adaptive immune defence system based on T- and B-cell recognition (Janeway at al., 1999; Vorup-Jansen et al., 1998). The recognition of targets for MBL binding is mediated by the C-type lectin domain. C-type CRDs are found in proteins with a widespread occurrence, both in phylogenetic and functional perspective. In the case of MBL, the CRD recognizes preferentially hexcos with equatorial 3- and 4-OH groups, such as mannose and N-acetyl glucoseamin while carbohydrates which do not fulfill this sterical requirement, such as galactose and D-fucose, are not bound (Weis et al., 1992).
The terminal CRDs are distributed in such a way that to allow for binding of all three domains target surfaces should present binding sites with a spacing of approximately 53 Å (Sheriff et al., 1994; Weis & Drickamer, 1994). This property of ‘pattern recognition’ may contribute further to the selectively binding of microbial surfaces. The carbohydrate selectivity is obviously an important aspect of the self/non-self discrimination by MBL and is probably mediated by the difference in prevalence of mannose and N-acetyl glucoseamin residues on microbial surfaces, one example being the high content of mannose in the cell wall of yeasts such as Saccharomyces cerevisiae and Candida albicans. Carbohydrate structures in glycosylation of mammalian proteins are usually completed with sialic acid, which prevents binding of MBL to these oligomeric carbohydrates and thus prevents MBL recognition of self surfaces. Also, the trimeric structure of each MBL subunit may be of importance for target recognition.
Several studies have been carried out on aspects of the structure and biosynthesis of MBL by use of in vitro synthesis systems. The structure of the CRD of rat MBL-A was resolved by crystallizing recombinant protein produced in E. coli (Weis of al., 1992), and the structure of rat MBL-C has likewise been resolved by use of recombinant material (Ng et al., 1996). More recently, the crystal structure of the trimer of CRDs assembled through expressing both the ‘neck-region’ and the CRD have confirmed the earlier studies on the ‘neck-region’ as bringing together the three chains by hydrogen bonds between the colled-coil α-helices (Sheriff et at., 1994; Webs & Drickamer, 1994). The CRDs and ‘neck-CRD’ fragments of other collectins have likewise been expressed in E. coli based systems without the requirement of refolding protocols to gain functional activity. Thus, the in vitro synthesis of MBL domains can both with regard to the functional properties of carbohydrate recognition and the structural property of trimerisation efficiently be carried out in prokaryotic or simple eukaryotic expression systems.
In vitro synthesis of complete collectins by recombinant technique have mainly been attempted by use of mammalian c 11 lines, e.g., CHO and COS cells, as host cells. Attempts to express MBL in insect cells only resulted in low m.w. MBL (Ma et al., 1996), the recombinant proteins almost entirely consisting of subunits, dimers, and trimers of the MBL subunit chains.
Recombinant synthesis of human MBL in mammalian cell lines have been reported in several studies. In a study on opsonic function i.e., ability to enhance uptake by macrophages, of MBL, Kuhlman et al. (1989) used rMBL produced in CHO cells, which showed the same opsonic activity, as natural MBL. Characterization of the rMBL with regard to structure and post-translational modifications was not carried out. Likewise, Schweinle et al. (1993) produced rMBL and a truncated form of rMBL lacking the collagenous tail by stable transfection of CHO cells. Activity of the recombinant proteins was measured by C3 deposition on Salmonella montevideo preincubated with MBL and diluted human serum depleted of antibodies against S. montevideo as complement source. Surprisingly, the activity of recombinant full-length MBL was as high as for natural MBL though analysis of iodinated rMBL by ultracentrifugation showed the majority of the protein to be low molecular weight material. Comparison of the CHO cell produced rMBL with natural MBL concerning size, either through SDS-PAGE, ultracentrifugation or GPC, was not included. In a study by Super et al. (1992), GPC profiles of rMBL produced in mouse Sp2/OAg14 hybridoma cells were presented indicating that the size distribution of rMBL purified from the culture supernatant by carbohydrate affinity chromatography was markedly different from rMBL isolated through anti-MBL antibody affinity chromatography with respect to size distribution.
Recently, Ohtani et al. (1999) published data on human rMBL produced in CHO cells, which gave a yield of 120 μg per mL of culture medium. This was obtained using a expression vector allowing for selection of transfectants by G418 resistance and subsequent gene amplification through selection with methionine sulfoximine (MSX). Functional activity concerning carbohydrate selectivity was identical to the natural protein whereas some differences in the ability to activate complement was found as measured through lysis of erythrocytes. Both the GPC and SDS-PAGE analysis showed the presence of higher molecular weight forms of MBL though the total size distribution was noted by the authors to differ significantly from MBL purified from plasma. Interestingly, the hydroxylation pattern was essentially identical to natural MBL ven in the absence of added ascorbic acid. Concerning the post-translation modifications, it should be noted from this study that proper hydroxylation of rMBL does not warrant a structure similar to natural MBL.
The human MBL locus is polymorphic with three known mutations located in the protein encoding region (Sumiya et al., 1991; Lipscombe et al., 1992a; 1992b; Madsen et at., 1994) and others affecting regulatory elements of the gene (Madsen et al., 1995). All mutations apparently leads to a significantly lower level of MBL in body fluids from affected individuals. The spread in MBL concentration as measured in serum is thus about three orders of magnitude, ranging from 2-5 μg MBL/mL to less than 10 ng MBL/mL in individuals homozygous for the mutations affecting protein coding regions. The finding of MBL deficiency being associated with recurrent infections in children which were diagnosed as suffering from an opsonic defect (Super et al., 1989; Sumiya et al., 1991) emphasized the correlation between insufficient MBL levels and reduction in the defence against micro-organisms. In addition to the interaction with micro-organisms, collectins have also been suggested to mediate anti-viral defence (Hartshorn et al., 1993; Malhotra et at., 1994). In vitro studies on interaction with human immunodeficiency virus (HIV) showed MBL to inhibit infection of CD4 positive T and U937 cells. Clinical studies also suggest a role for MBL as a first-line defence against HIV. The period of time from the onset of symptoms of AIDS until the terminal stage seem to differ between MBL-deficient patients and patients with normal MBL levels. Also, the susceptibility to contract the infection appears to be significantly higher among MBL-deficient individuals as MBL deficiency occurs more frequently among HIV infected patients than healthy controls (Nielsen et al., 1995; Garred et al., 1997a). Hereditary complement deficiencies contribute to development of systemic lupus erythematosus (SLE) as have been shown for cases of C1, C2, and C4 deficiency (reviewed by Tan & Arnett, 1998). Several studies have suggested MBL deficiency likewise to be a risk factor in development of SLE (Davies et al., 1995; Lau et al., 1996; Ip et al., 1998) although the role of MBL is not well defined. One suggestion, following explanations for the other complement deficiencies as causative agents in SLE, argues that the defect in complement fixation due to MBL deficiency leads to poor immune complex clearance (Ip et al., 1998), thus pointing to MBL as a participant in maintaining homeostasis. Other clinical evidence suggests MBL to play an important role in reproductive biology. Recent reports show association between recurrent miscarriages and the MBL level (Kllpatrick et al., 1995; Christiansen et al., 1999).
A strategy for treating symptomatic MBL insufficient individuals aiming at reconstituting the MBL pathway has only been described in two studies. An opsonic detect observed among some children, at the time only characterized by the clinical manifestation of frequent infections, was treated by administering plasma (Soothill & Harvey 1976) while, in a more recent study, plasma-derived MBL was injected into a two-year-old girl (Valdimarsson et al., 1998). In both studies a health improvement was reported though the small number of patients involved in these studies obviously limits conclusions with regard to MBL as therapeutic agent. No clinical studies have been conducted with rMBL. A study by Ma et al. (1999) showed an antitumor activity of rMBL in mice mediated by a virus expression system.
As appears from the above, several attempts have been made to produce rMBL in in vitro systems in a form either identical to or with a high degree of resemblance to the natural protein. However, significant limitations have been demonstrated in the ability of the presently known in vitro synthesis systems to produce such rMBL.